AMINO ACID METABOLISM--ENZYME BIOGENESIS AND MUTATION

氨基酸代谢——酶的生物发生和突变

• 批准号: 2292198
• 负责人: FRANTISEK KALOUSEK
• 金额: $ 2.43万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2292198
• 关键词: Escherichia coli; Neurospora; X ray crystallography; active sites; adenosine triphosphate; aminoacid metabolism; carboxyltransferase /carbamoyltransferase; enzyme biosynthesis; enzyme mechanism; enzyme structure; enzyme substrate; exopeptidase; gene mutation; hydrolysis; intermolecular interaction; mitochondria; molecular chaperones; molecular cloning; protein biosynthesis; protein folding; protein purification; protein reconstitution; protein sequence; site directed mutagenesis; yeasts

The research proposed in this application reflects a continuing interest in the mechanisms by which nuclearly coded mitochondrial proteins are proteolytically processed post-translocationally to produce mature, active enzymes. This process is central to the maintenance and propagation of mitochondria, and, hence, to cellular metabolism and homeostasis. This pathway must be understood in order to account for certain aspects of human metabolic diseases and to rationally design strategies for their somatic correction. The specific aims are: 1) to express in E. coli both subunits of yeast mitochondrial processing peptidase (MPP) and reconstitute its activity; and 2) to carry out site-directed mutagenesis of the active site of yeast MPP and its putative substrate binding site. The proposed research will complement and extend that carried out on rat MPP, the homologous protease from a very dissimilar organism. Site- directed mutagenesis, heterologous expression, purification of MPP, and in vitro assay of its proteolytic activity, metal-ion binding, and substrate binding will yield data on the roles of conserved residues and regions in various aspects of the enzymatic mechanism. Because the homology between rat and yeast MPP is relatively high and the general characteristics of the enzymes are similar, it will be possible to compare and contrast the effects of the same mutations in two homologous enzymes to provide additional insights into the mechanism of the overall reaction and the importance of the inter-species variations for the biological role of this essential enzyme.

在这项申请中提出的研究反映了持续的兴趣 在细胞核编码线粒体蛋白的机制中， 易位后蛋白水解加工以产生成熟的、活性的 内切酶这一过程是维护和传播 线粒体，并因此影响细胞代谢和体内平衡。这 必须了解途径，以说明某些方面的 人类代谢疾病，并合理设计其策略， 躯体矫正具体目标是：1）在E.大肠杆菌 酵母线粒体加工肽酶（MPP）的亚基和 重建其活性; 2）进行定点突变 酵母MPP的活性位点和其假定的底物结合位点。 拟议的研究将补充和扩展对大鼠进行的研究。 MPP，来自非常不同的生物体的同源蛋白酶。研究中心- 定向诱变，异源表达，MPP的纯化，以及 其蛋白水解活性、金属离子结合和底物的体外测定 结合将产生关于保守残基和区域在 酶机制的各个方面。因为它们之间的同源性 大鼠和酵母菌MPP相对较高，一般特点是 酶是相似的，将有可能比较和对比 在两种同源酶中的相同突变的影响， 对整个反应机制的进一步了解和 物种间变异对生物学作用的重要性 必需酶